The present invention relates to a UBP1 protease mutant and the sequence coding it, their application as well as products and methods for their production. The present invention is used in the production of recombinant proteins, particularly on an industrial scale.
Ubiquitin is a protein commonly expressed in eukaryotes. It has been shown that it is a useful carrier for heterologous proteins obtained through expression in Escherichia coli (R. Baker, Current Opinion in Biotechnology 1996, 7:541-546). Ubiquitin is composed of 76 amino-acid residues, with a combined molecular mass of 8.8 kDa. This protein is an element of the universal protein modification system. Ubiquitination is involved in almost all metabolic processes, from cell division to its death. Ubiquitin is involved in the regulation of gene expression, DNA repair, and it influences chromatin activity. It takes part in oncogenesis. It also plays a pivotal role in the proteolysis of regulatory proteins with short half-lives, and proteins with longer half lives as well, which must be removed from a cell for various reasons.
Protein ubiquitination does not occur in bacteria. It has been shown that proteins fused to ubiquitin undergo greater expression in E. coli, and are more stable. Crystallographic analysis of ubiquitin using nuclear magnetic resonance demonstrated that both in solid state and in aqueous solution ubiquitin maintains a dense, globular structure (S. Vijay-Kumar, C. Bugg, W. Cook, J. Mol. Biol. 1987, 194:531-544). The hydrophobic core of ubiquitin is composed of five parallel lengths of the peptide chain, held together with regularly spaced hydrogen bonds, forming a so-called β-pleated sheet. Its surface edges are joined with short chain lengths, coiled into 3.5 turns of an α-helix. Such a structure gives ubiquitin an uncommon resistance to high temperatures, a wide range of pH and polarity changes in the environment (Harding M M, Williams D H, Woolfson D N Biochemistry 1991, 30:3120-3128).
The UBP1 protease is an enzyme isolated from yeast, which severs ubiquitin from a protein located at its C-end. The enzyme was described in 1991 (J. Tobias, A. Varshavsky, J. Biol. Chem. 1991, 266; 12021-12028) and is a subject of patent application WO91/17245 (European Patent EP 531 404). Its activity has been studied, and its culture conditions have been described in E. coli. In accordance to the contents of the description, it is a cysteine protease, which binds ubiquitin with an ester bond. UBP1 is made up of an 809 amino-acid chain. The enzymatic activity depends on its ability to sever the ubiquitin peptide from the polypeptide found at its C-end, regardless of the amino-acid sequence at the N-end of the ubiquitin.
Application WO93/09235 describes other yeast proteins belonging to the same protease family, namely UBP2 and UBP3. This proteins show a similar activity (see also U.S. Pat. No. 5,494,818, U.S. Pat. No. 5,212,058, U.S. Pat. No. 5,683,904).
No improved mutants of UBP1 have been shown to date.
Expression systems are known, which yield fusion proteins composed of ubiquitin or its derivative and a polypeptide of interest. These then apply an enzyme which removes the ubiquitin (ie. UBP1), and recover the protein of interest (for examples see U.S. Pat. No. 5,132,213, U.S. Pat. No. 6,018,102). Such a method contains many advantages, encompassing among others an improvement in the quality and yield efficiency of the protein, and a simplification of the purification process of the protein, a significant feature for the industrial production of recombinant proteins (for an example see WO03/010204). Using an enzyme which removes ubiquitin along with appropriately designed fusion proteins, one may also obtain N-modified polypeptides (for example U.S. Pat. No. 5,847,097).
The application of an enzyme which removes ubiquitin in technological processes requires large amounts of this enzyme. Known methods, however, are not conducive to the efficient expression of said enzyme, and significantly limit the possibilities of its application, particularly in industrial processes.